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Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification. | LitMetric

Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification.

Protein Eng Des Sel

Division of Experimental Therapeutics, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA Genecology Research Centre, Inflammation and Healing Research Cluster, School of Science and Engineering, University of the Sunshine Coast, Queensland 4556, Australia

Published: November 2015

AI Article Synopsis

Article Abstract

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

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Source
http://dx.doi.org/10.1093/protein/gzv036DOI Listing

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